In vertebrates, synthesis and release of the two gonadotrophic hormones, luteinizing hormone (LH) and follicle stimulating hormone (FSH), are regulated by a polypeptide referred to as Gonadotropin releasing hormone (GnRH) (formerly designated LHRH). Accordingly, one approach to fertility control in an animal population is to reduce the levels of GnRH, such as by immunization against GnRH, which effects a reduction in the levels of LH and FSH and the concomitant disruption of estrous cycles and spermatogenesis. See e.g., Adams et al., J. Anim. Sci. (1990) 68:2793-2802.
In particular, early studies of the GnRH molecule have shown that it is possible to raise antisera in response to repeated injections of synthetic GnRH peptides (Arimura et al., Endocrinology (1973) 93(5):1092-1103). Further, antibodies to GnRH have been raised in a number of species by chemical conjugation of GnRH to a suitable carrier and administration of the conjugate in an appropriate adjuvant (Carelli et al., Proc. Natl. Acad. Sci. (1982) 79:5392-5395). Recombinant fusion proteins comprising GnRH or GnRH-analogues have also been described for use in peptide vaccines for the immunological castration or inhibition of reproductive function of various domesticated and farm animals (Meloen et al., Vaccine (1994) 12(8):741-746; Hoskinson et al., Aust. J. Biotechnol. (1990) 4:166-170; and International Publication Nos. WO 92/19746, published Nov. 12, 1992; WO 91/02799, published Mar. 7, 1991; WO 90/11298, published Oct. 4, 1990 and WO 86/07383, published Dec. 18, 1986).
However, attempts have fallen short of providing adequate immunological sterilization products due to the poor immunogenicity of GnRH peptides and due to the fact that chemical conjugation protocols are difficult to control, rendering substantially heterogenous and poorly-defined GnRH conjugates. Further, peptide vaccines based on GnRH have met with limited success in providing uniform effects on individual animal subjects even after repeated vaccination. In this regard, prior GnRH constructs have failed to provide a uniformly successful immunological sterilization vaccine product due to the fact that GnRH is a small, "self" molecule that is not normally recognized by a subject's immune system, rendering the molecule poorly immunogenic and inherently unable to induce a significant immune response against endogenous GnRH.
It is generally recognized that the immunogenicity of viral antigens, small proteins or endogenous substances may be significantly increased by producing immunogenic forms of those molecules comprising multiple copies of selected epitopes. In this regard, constructs based on two or four repeats of peptides 9-21 of herpes simplex virus type 1 glycoprotein D (Ploeg et al., J. Immuno. Methods (1989) 124:211-217), two to six repeats of the antigenic circumsporozoite tetrapeptide NPNA of Plasmodium falciparum (Lowell et al., Science (1988) 240:800-802), two or four copies of the major immunogenic site of VP1 of foot-and-mouth disease virus (Broekhuijsen et al., J. gen. Virol. (1987) 68:3137-3143) and tandem repeats of a GnRH-like polypeptide (Meloen et al., Vaccine (1994) 12(8):741-746), have been shown to be effective in increasing the immunogenicity of those molecules.
Furthermore, small proteins or endogenous substances may also be conjugated to a suitable carrier in order to elicit a significant immune response in a challenged host. Suitable carriers are generally polypeptides which include antigenic regions of a protein derived from an infectious material such as a viral surface protein, or a carrier peptide sequence. These carriers serve to non-specifically stimulate T helper cell activity and to help direct antigen to antigen presenting cells for processing and presentation of the peptide at the cell surface in association with molecules of the major histocompatibility complex (MHC).
Several carrier systems have been developed for this purpose. For example, small peptide antigens are often coupled to protein carriers such as keyhole limpet haemocyanin (Bittle et al., Nature (1982) 298:30-33), tetanus toxoid (Muller et al., Proc. Natl. Acad. Sci. U.S.A. (1982) 79:569-573), ovalbumin, and sperm whale myoglobin, to produce an immune response. These coupling reactions typically result in the incorporation of several moles of peptide antigen per mole of carrier protein. Although presentation of the peptide antigen in multiple copies generally enhances immunogenicity, carriers may elicit strong immunity not relevant to the peptide antigen and this may inhibit the immune response to the peptide vaccine on secondary immunization (Schutze et al, J. Immun. (1985) 135:2319-2322).
Antigen delivery systems have also been based on particulate carriers. For example, preformed particles have been used as platforms onto which antigens can be coupled and incorporated. Systems based on proteosomes (Lowell et al., Science (1988) 240:800-802), immune stimulatory complexes (Morein et al., Nature (1984) 308:457-460), and viral particles such as HBsAg (Neurath et al., Mol. Immunol. (1989) 26:53-62) and rotavirus inner capsid protein (Redmond et al., Mol. Immunol. (1991) 28:269-278) have been developed.
Carrier systems have also been devised using recombinantly produced chimeric proteins that self assemble into particles. For example, the yeast retrotransposon, Ty, encodes a series of proteins that assemble into virus like particles (Ty-VLPs; Kingsman, S. M., and A. J. Kingsman Vacc. (1988) 6:304-306). Foreign genes have been inserted into the TyA gene and expressed in yeast as a fusion protein. The fusion protein retains the capacity to self assemble into particles of uniform size.
Other chimeric protein particles have been examined such as HBsAg, (Valenzuela et al., Bio/Technol. (1985) 3:323-326; U.S. Pat. No. 4,722,840; Delpeyroux et al., Science (1986) 233:472-475), Hepatitis B core antigen (Clarke et al., Vaccines 88 (Ed. H. Ginsberg, et al., 1988) pp. 127-131), Poliovirus (Burke et al., Nature (1988) 332:81-82), and Tobacco Mosaic Virus (Haynes et al., Bio/Technol. (1986) 4:637-641). However, these carriers are restricted in their usefulness by virtue of the limited size of the active agent which may be inserted into the structural protein without interfering with particle assembly.
Finally, chimeric systems have been devised using a Pasteurella haemolytica leukotoxin (LKT) polypeptide fused to a selected antigen. See, e.g., International Publication Nos. WO 93/08290, published Apr. 29, 1993 and WO 92/03558, published Mar. 5, 1992, as well as U.S. Pat. Nos. 5,238,823 and 5,273,889. Inclusion of a LKT carrier portion in a peptide antigen chimera supplies enhanced immunogenicity to the chimera by providing T-cell epitopes having broad species reactivity, thereby eliciting a T-cell dependent immune response in immunized subjects. In this regard, inducement of adequate T-cell help is essential in the generation of an immune response to the peptide antigen portion of the chimera, particularly where the antigen is an endogenous molecule. However, the use of a leukotoxin polypeptide carrier in combination with multiple epitopes of the GnRH peptide has not heretofore been described.